Large expansions (>200 CGG repeats; full mutation) of a CGG-repeat in the FMR1 gene cause the leading heritable form of intellectual disability (fragile X syndrome) and are associated with the leading known single- gene form of autism. Smaller expansions (55-200 CGG repeats; premutation) give rise to one of the most common known single-gene neurodegenerative disorders, fragile X-associated tremor/ataxia syndrome (FXTAS), and also result in the leading single-gene form of primary ovarian failure. Therefore, the consequences of CGG-repeat expansions are of great societal impact, with over one-hundred thousand individuals in the United States affected by one or more of the fragile X-associated disorders. In both premutation and full-mutation disorders, disease pathogenesis is due to altered levels of expression - reduced/absent protein for fragile X syndrome and elevated mRNA for FXTAS. In the proposed research, we will exploit a novel method for single-molecule genomic (SMRT) sequencing, coupled with the generation of single-allele fibroblast sub-clones, to determine at the sequence-level the CGG-repeat lengths and complexity (i.e., size mosaicism) of expanded CGG-repeat FMR1 alleles (Specific Aim 1) and to define the dependence of FMR1 protein (FMRP) levels on CGG-repeat length and to identify at least some of the factors that control FMRP expression (Specific Aim 2). In addition, we will further define the basis for increased mRNA levels for FMR1 alleles within the premutation range (Specific Aim 3), and will exploit our earlier observation that the start of transcription shifts upstream as the CGG-repeat expands, with much of the excess mRNA being produced by the additional, upstream initiator. We hypothesize that targeting the region of the mRNA between the normal and upstream initiators will effectively lower mRNA levels toward the normal range, without excess reduction. This proposal, representing another potential therapeutic approach to FXTAS, will be evaluated as part of Specific Aim 3.